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a) in a recombinant DNA cloning experiment, how can we determinewether DNA Fragments of interest have been incorporated intoplasmids and, once host cells are transformed, which cells containrecombinant DNA?

b) when using DNA Libraries to clone genes, what combination oftechniques are used to identify a particular gene of interest?

c) what steps make PCR a chain a chain reaction that can producemillions of copies of a specific DNA molecule in a matter of hourswithout using host cells?

d) how has DNA sequencing technology evolved in response to theemerging needs of genome scientists?

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Nelly Stracke
Nelly StrackeLv2
28 Sep 2019

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Related questions

1.Whati is Genetic Technology?What is bacterial transformation?
What is recombinant DNA technology?What is a vector?What is found in a typical plasmid?How can DNA sequenced use the dideoxy chain termination method? Describe the polymerase chain reaction (PCR). What are some advantages and limitations of this procedure?What is a cDNA? How are cDNAs and DNA microarrays used to study gene expression?Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology,Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA moleculOutline the procedures for cloning a eukaryotic gene in a bacterial plasmid.Describe techniques that allow identification of recombinant cells that have taken up a gene of interest.How can you use the lacZ gene to facilitate cloning ?Distinguish between genomic and cDNA libraries.Define genomics and proteomics.Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene.How does dideoxy sequencing work? What is the significance of using dideoxy nucleotides?What are BACs, YACs, and contigs? What are some practical applications of DNA technology?What are the ways in which organisms can be genetically modified?

ANSWER THE FOLLOWING QUESTIONS(COULD BE MORE THAN ONEANSWER)

19) Describe the roles of M13 genes and their proteins:

a) Genes 1, 4 and 11 produces proteins required for assemblingthe the phage.

b)Genes 3.6,7,8 and 9 produces capsid proteins.

c)Gene 2 nicks the circular phage ssDNA to make it doublestranded by DNA polymerase

d)Gene 5 produces single strand binding proteins and gene 10protein makes M13 SSDNA circular.

e)Protein five bound single stranded circular M13 moved to theE. coli membranne and is released out coated will proteins 3, 6, 7,8 and 9 .

2) What is a phagemid and how does it work?

a)it is a recombinant DNA with a filamentous phage genome and aplasmid genome.

b)it carries two origins of replication: eg., oir f1 and oriColE1

c)May carry a gene for selection: e.g. Ampr gene.

d)May carry a marker gene; e.g, lacZ gene with promoter andregulator

e)May contain MCS, as is pUC18.

3)For storing functional gene for future applications you willneed the following:

a)A vector DNA

b) A cloning site in the vector matching with the ends of a geneto be cloned

c)A host organism capable to hold the recombined vector.

d)A marker gene present in the recombinant vector

e)A functional gene of interest.

24)identify which of the following is true for thebacteriophage lambda?

a)It has two major body parts, the head and the tail.

b)The genome is approximately 49 thousand bases long.

c) It has a linear dsDNA with single stranded 12 bases long costerminals at both

d) The single stranded lambda DNA cos terminals appear to be asfollows: (a) GGGCGGCGACCT and (b)CCCGCCGCTGGA

e)it can accept 7-10 kb long foreign DNA replacing itsnon-essential b2 region.

29) For developing a recombinant lambda DNA with a gene ofinterest and expressing the gene in a host you have to take thefollowing steps:

a)Remove the b2 region from lambda DNA using a restrictionenzyme

b)digest the gene of interest, outside its stop and startcodons, with the same restriction enzyme.

c)Recombine the lambda DNA digest with the digested gene ofinterest carrying complementary overhangs with ligase.

d)Form lambda particles by adding head proteins, tail proteinsand packaging enzynes to the recombined lambda DNA.

e)Add the packaged lambda particles to Agrobacetrium tumefaciensand incubate them in culture.

f)Add the packaged lambda particles to laboratory strains ofEscherichia coli and incubate them in culture.

32)The amino acids coded by our genes differ by thefollowing:

a)Polarity

b) Acidity

c)Basicity

d)Aroma

e)Hydrophobicity

38)What is an M13 phage and what are its specialty?

a)They are rod shaped Escherichia coli bacteriophage.

b)ts genome is made of a single stranded circular DNA.

c)Its DNA is 6400 nucloetides long coding for elevenproteins.

d)After infecting a bacterial cell it starts replicating in 10minutes producing 1,000 phages in an hour

e) soon its kills the host Escherichia coli and gets out to theenvironment.

40)You have a gene with BamH cut sites out side its start andthe stop codons. You can use the following procedures to prepare aclone of the gene in pBR322 and select the transformed E.Coli cellscarrying pBR322, except:

a) Digest both the gene and the plasmid pR322 with BamH1

b) Ligate the fragments using T4 DNA ligase.

c)Transform E coli with the reconstructed plasmid

d)Select the cells transformed cells grow in Lb-agar-ampicilinplates

e)Select the cells transformed cells grow inLB-agar-tetracycline plates

2.Conclusion:

What have you learned from your study?

Interpret your data/ results:

Do your results support of disprove your hypothesis?

Compare and contrast your result with those of previous research.

Significance of the results: “Big Picture”

What is the global impact of your results?

What questions remain unanswered?

What kinds of experiments can come out of your study?

i have lab for biology and i need help with the Lab Report

the lab name is Bacterial Transformation Lab

1. Introduction:

Introduce the background and the nature of the problem your experiment is investigating.

Summarize the current knowledge of your subject.

Use examples of other research that have examined the same or a similar topic as your experiments.

Ending your introduction:

State your hypothesis

State your predictions

Give a brief statement about how you tested your hypothesis.

please help me with it is important for my lab class tomorrow

that is the lab i put the info please help me for the intro and for number 2 please be clear please

i would be happy if you help me it is due tommorrow i realy need your help on the intro and number 2 please be clear and show clear work hope you do it because it is due tomorrow

Bacterial Transformation Lab

Genetic engineering is one of the newest applications of basic knowledge in biology. It involves the isolation of specific genes from one organism and the insertion of those genes into a second organism of the same, or even different, species. Remember that a gene is a piece of DNA which codes for a product, usually a protein, which gives an organism a particular trait. The development of these techniques has generated considerable excitement and answered many questions, but it has also raised questions. Many scientists and others see genetic engineering as offering opportunities to make life better, for example, by exploring how genes are regulated, by curing diseases of genetic origin, by adding desirable traits to agricultural crops such as the ability to fix nitrogen in corn and pest resistance in cotton, and by genetically altering bacteria with genes enabling them to digest oil spills.

For almost 100 years, it has been possible to isolate DNA from cells, but only in the last 40 years or so has its function been understood. Your textbook discusses the work of such investigators as Fred Griffith and Oswald Avery, who demonstrated that if DNA was isolated from a pneumonia-causing strain of bacteria and added to cultures of nonvirulent bacteria, the recipient cells were transformed into virulent types that caused pneumonia. These transformation experiments were the first to show that genes could be artificially transferred in the laboratory. Since the time of those experiments (Avery's work was done in the 1940s), our knowledge of gene structure, function and control has increased astronomically. The application of this knowledge has created the field of genetic engineering.

Modern genetic engineering techniques depend on three critical factors:

A suitable host organism in which to insert the foreign gene;

A vector to carry foreign genes into the host; and

A means of isolating the host cells that have taken up the foreign gene.

The host organism

The most commonly used host organism is the gut bacterium Escherichia coli (E. coli). Its chromosome is a single large circular DNA molecule, called the genomic DNA, containing about 4 x 106 base pairs. Not every cell will take up foreign DNA when exposed to it, but E. coli grows rapidly, dividing in less than 20 minutes, so that a single cell can give rise to a billion genetically identical descendants in a matter of 10 hours. Thus, a few transformed cells can provide a large number of offspring within a day. The strain we are using is K-12.

The vector

A vector is a DNA molecule used as a vehicle to carry genes from one organism to another. This DNA molecule can be modified by gene-splicing techniques using restriction enzymes and ligases so that it carries the genes of interest (essentially a cut-and-paste operation). Sometimes viruses are used as vectors, but for E. coli, plasmids are often used. A plasmid is a small circular DNA molecule containing 1000 to 200,000 base pairs. Plasmids exist naturally in many strains of E. coli and other bacteria. Plasmids replicate as the cell grows, independent of replication of the genomic DNA. These plasmids are transmitted to each of the product cells during cell division. Plasmids carrying genetic information that is beneficial to the host cell are maintained in a given population by natural selection. Plasmids frequently carry genes that confer antibiotic resistance, an obvious benefit.

When E. coli cells are exposed to plasmids or any other foreign DNA, some of the cells, called competent cells, will take up the DNA. We can induce cells to become competent by treating them with divalent cations, such as Ca++, followed by a brief exposure to high temperature, called a heat shock treatment. Exactly how this treatment causes cells to become competent is not understood, but it is described as making the membranes "leaky" so that large DNA molecules (like plasmids), which would not normally pass through the cell membrane, can enter the cell. This treatment does not make all cells competent, but it greatly increases the number of competent cells. Once in a cell, the plasmids replicate and are passed to the product cells during cell division.

The selective medium

The last requirement for genetic engineering is a means of isolating host cells that have taken up the gene of interest. The techniques used depend on the growth requirements of the host organism. For example, each species of bacteria has particular nutrient requirements. The composition of the growth medium (agar or broth) can be modified to allow growth of only certain bacteria -- for example, by adding or removing nutrients or other chemicals from the medium mixture. This modified medium is called a selective medium, because it selects for the growth of only some species. By introducing an antibiotic such as ampicillin into the medium in which the bacteria are grown, you will select for growth of only those bacteria carrying the antibiotic-resistance gene on newly acquired plasmids. In a matter of days, billions of antibiotic-resistant cells, all with the plasmid, can be grown.

Genetic engineers use a modification of this technique to insert a foreign gene into E. coli. In simplified terms, the plasmid (already carrying an ampicillin resistance gene) is modified by inserting the gene of interest. The gene of interest has been isolated from another source; for example, the gene for human insulin has been isolated from the human genome. The modified plasmid, carrying the inserted gene, is then mixed with competent E. coli and the bacteria are placed in a medium containing ampicillin. Only those cells that have the plasmid will grow in the medium, and they are also the cells carrying the insulin gene (in their plasmids). These selected cells can now be used to manufacture insulin, which can be used for insulin therapy by diabetics.

The genes

In the following experiment, you will insert a plasmid into E. coli. The plasmid you will use carries the gene bla, which codes for a protein (beta-lactamase) that is secreted by the bacteria, inactivating the ampicillin in the agar and allowing for bacterial growth. The plasmid also carries an indicator gene. The indicator gene we are using was isolated from a bioluminescent jellyfish, Aequorea victoria. It codes for Green Fluorescent Protein (GFP).

The GFP gene has been modified to include a regulator system that can be used to control the expression of the gene. Gene expression in all organisms is carefully regulated to allow for adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the sugar arabinose is both an energy source and a carbon source for bacteria. The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But, when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off.

Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene GFP. When bacteria with this plasmid are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow green in UV light. When arabinose is absent form the growth medium, the GFP gene remains turned off and the colonies appear white. This is an example of the central molecular framework of biology in action: DNA ? RNA ? protein ? trait.

In summary, you will render E. coli cells competent to take up plasmid DNA. The plasmid carries two identifiable phenotypic markers:

The beta-lactamase or bla gene codes for resistance to the antibiotic ampicillin. We will use this to select for bacteria that have taken up the plasmid.

The pGLO gene codes for Green Fluorescent Protein (GFP), which is switched “ON” if arabinose is present.

Safety Procedure

For your own safety and the safety of others, it is essential that you follow all instructions about handling bacteria and disposal of materials. When you finish work for the day, swab your table with disinfectant. WASH YOUR HANDS thoroughly with soap before leaving the lab for any reason, and at the end of the day after you have cleaned your table. Report any spills to your instructor.

List of Materials

Prepared agar plates (2 LB, 2 LB/amp, 1 LB/amp/ara)

Transformation solution (50 mM CaCl2, pH 7.4)

LB broth

1 pack of inoculation loops

Sterile transfer pipets

1 foam microtube holder w/ tubes

E. coli starter plate

1 red biohazard bag for loop disposal (common use – Loops/pipets only - NO PAPER!!!)

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